Widespread Use of the Nitrification Inhibitor Nitrapyrin: Assessing Benefits and Costs to Agriculture, Ecosystems, and Environmental Health.

Agricultural production and associated applications of nitrogen (N) fertilizers have increased dramatically in the last century, and current projections to 2050 show that demands will continue to increase as the human population grows. Applied in both organic and inorganic fertilizer forms, N is an essential nutrient in crop productivity. Increased fertilizer applications, however, create the potential for more N loss before plant uptake. One strategy for minimizing N loss is the use of enhanced efficiency fertilizers, fortified with a nitrification inhibitor, such as nitrapyrin. In soils and water, nitrapyrin inhibits the activity of ammonia monooxygenase, a microbial enzyme that catalyzes the first step of nitrification from ammonium to nitrite. Potential benefits of using nitrification inhibitors range from reduced nitrate leaching and nitrous oxide emissions to increased crop yield. The extent of these benefits, however, depends on environmental conditions and management practices. Thus, such benefits are not always realized. Additionally, nitrapyrin has been shown to transport off-field, and it is unknown what effects environmental nitrapyrin could have on nontarget organisms and the ecological nitrogen cycle. Here, we review the agronomic and environmental benefits and costs of nitrapyrin use and present a series of research questions and considerations to be addressed with future nitrification inhibitor research.

Prevalence of Eight Phthalate Monoesters in Water from the Okavango Delta, Northern Botswana.

Phthalate diesters are used in personal care products, plastics, and pesticides, resulting in widespread human and wildlife exposure. Phthalate diesters leach out of these products and ultimately enter biological systems where they are quickly metabolized to phthalate monoesters and glucuronides. As such, phthalate monoesters can serve as indicators of anthropogenic activity in wilderness areas. The Okavango Delta, an inland seasonal wetland covering 5000-12,000 km2 in Botswana, provides fresh water to many species of birds, fish, reptiles, and large mammals. Water samples (N = 46) were taken from across the Okavango water system, extracted, and analyzed for eight different phthalate monoesters using liquid chromatography and isotope dilution mass spectrometry. Seven of eight phthalate monoesters were detected from the low ng/L to low µg/L levels. Phthalate monoesters were found in samples from all five sampling regions. Sources of these contaminants are unknown, but their presence indicates encroachment of human activity on the Okavango Delta.

Reproductive endocrinology of environmental nitrate.

Nitrate is a widespread contaminant of aquatic ecosystems and drinking water. It is also broadly active in organismal physiology, and as such, has the potential to both enhance and disrupt normal physiological function. In animals, nitrate is a proposed endocrine disrupter that is converted in vivo to nitrite and nitric oxide. Nitric oxide, in particular, is a potent cell signaling molecule that participates in diverse biological pathways and events. Here, we review in vivo nitrate cycling and downstream mechanistic physiology, with an emphasis on reproductive outcomes. However, in many cases, the research produces contradictory results, in part because there is good evidence that nitrate follows a non-monotonic dose-response curve. This conundrum highlights an array of opportunities for scientists from different fields to collaborate for a full understanding of nitrate physiology. Opposing conclusions are especially likely when in vivo/in vitro, long term/short term, high dose/low dose, or hypoxia/normoxia studies are compared. We conclude that in vivo studies are most appropriate for testing an organism's integrated endocrine response to nitrate. Based on the limited available studies, there is a generalized trend that shorter term studies (less than 1 month) or studies involving low doses (≤5 mg/L NO3-N) cause steroid hormone levels to decline. Studies that last more than a month and/or involve higher, but still environmentally relevant, exposures (>50-100 mg/L NO3-N) cause steroid hormone levels to increase. Very high nitrate doses (>500 mg/L NO3-N) are cytotoxic in many species. Hypoxia and acidity are likely to intensify the effects of nitrate. For study design, degree of study animal reproductive maturity or activity is important, with immature/reproductively quiescent animals responding to nitrate differently, compared with reproductively active animals. A detailed table of studies is presented.

Nitrate induces a type 1 diabetic profile in alligator hatchlings.

Type 1 diabetes (T1D) is a chronic autoimmune disease that affects 1 in 300 children by age 18. T1D is caused by inflammation-induced loss of insulin-producing pancreatic beta cells, leading to high blood glucose and a host of downstream complications. Although multiple genes are associated with T1D risk, only 5% of genetically susceptible individuals actually develop clinical disease. Moreover, a growing number of T1D cases occur in geographic clusters and among children with low risk genotypes. These observations suggest that environmental factors contribute to T1D etiology. One potential factor, supported primarily by epidemiological studies, is the presence of nitrate and nitrite in drinking water. To test this hypothesis, female hatchling alligators were exposed to environmentally relevant concentrations of nitrate in their tank water (reference, 10mg/L, or 100mg/L NO3-N) from hatch through 5 weeks or 5 months of age. At each time point, endpoints related to T1D were investigated: plasma levels of glucose, triglycerides, testosterone, estradiol, and thyroxine; pancreas, fat body, and thyroid weights; weight gain or loss; presence of immune cells in the pancreas; and pancreatic beta cell number, assessed by antibody staining of nkx6.1 protein. Internal dosing of nitrate was confirmed by measuring plasma and urine nitrate levels and whole blood methemoglobin. Cluster analysis indicated that high nitrate exposure (most animals exposed to 100mg/L NO3-N and one alligator exposed to 10mg/L NO3-N) induced a profile of endpoints consistent with early T1D that could be detected after 5 weeks and was more strongly present after 5 months. Our study supports epidemiological data correlating elevated nitrate with T1D onset in humans, and highlights nitrate as a possible environmental contributor to the etiology of T1D, possibly through its role as a nitric oxide precursor.

Environmentally relevant concentrations of nitrate increase plasma testosterone concentrations in female American alligators (Alligator mississippiensis).

Anthropogenic nitrogen is a ubiquitous environmental contaminant that is contributing to the degradation of freshwater, estuarine, and coastal ecosystems worldwide. The effects of environmental nitrate, a principal form of nitrogen, on the health of aquatic life is of increasing concern. We exposed female American alligators to three concentrations of nitrate (0.7, 10 and 100mg/L NO3-N) for a duration of five weeks and five months from hatch. We assessed growth, plasma sex steroid and thyroid hormone concentrations, and transcription levels of key genes involved in steroidogenesis (StAR, 3ß-HSD, and P450scc) and hepatic clearance (Cyp1a, Cyp3a). Exposure to 100mg/L NO3-N for both five weeks and five months resulted in significantly increased plasma testosterone (T) concentrations compared with alligators in the reference treatment. No differences in 17ß-estradiol, progesterone, or thyroid hormones were observed, nor were there differences in alligator weight or the mRNA abundance of steroidogenic or hepatic genes. Plasma and urinary nitrate concentrations increased with increasing nitrate treatment levels, although relative plasma concentrations of nitrate were significantly lower in five month, versus five week old animals, possibly due to improved kidney function in older animals. These results indicate that environmentally relevant concentrations of nitrate can increase circulating concentrations of T in young female alligators.

Integrative and comparative reproductive biology: From alligators to xenobiotics.

Dr. Louis J. Guillette Jr. thought of himself as a reproductive biologist. However, his interest in reproductive biology transcended organ systems, life history stages, species, and environmental contexts. His integrative and collaborative nature led to diverse and fascinating research projects conducted all over the world. He doesn't leave us with a single legacy. Instead, he entrusts us with several. The purpose of this review is to highlight those legacies, in both breadth and diversity, and to illustrate Dr. Guillette's grand contributions to the field of reproductive biology. He has challenged the field to reconsider how we think about our data, championed development of novel and innovative techniques to measure endocrine function, helped define the field of endocrine disruption, and lead projects to characterize new endocrine disrupting chemicals. He significantly influenced our understanding of evolution, and took bold and important steps to translate all that he has learned into advances in human reproductive health. We hope that after reading this manuscript our audience will appreciate and continue Dr. Guillette's practice of open-minded and passionate collaboration to understand the basic mechanisms driving reproductive physiology and to ultimately apply those findings to protect and improve wildlife and human health.

Seasonal reproduction of male Gambusia holbrooki (eastern mosquitofish) from two Florida lakes.

Sixteen monthly collections of adult male Gambusia holbrooki (eastern mosquitofish) were obtained from two lakes in central Florida, USA. Lake Woodruff and Lake Apopka are 36 miles apart, but differ in several environmental parameters. Compared with Lake Woodruff, Lake Apopka is warmer, more shallow in sampling areas (particularly during drought conditions; approximately 15-90 cm in Lake Apopka versus 60-120 cm in Lake Woodruff), more turbid, and more heavily contaminated with nutrients and industrial and agricultural chemicals. Here, we present detailed information on seasonal reproduction patterns in mosquitofish in their native range and compare patterns between fish from the two lakes. Male mosquitofish were reproductively active from spring through fall. Spermatogenesis, which is regulated in part by 11-ketotestosterone, ceased in October, and fish stored spermatozoa through the winter for immediate fertilization of offspring in the spring. Compared with Lake Woodruff, fish from Lake Apopka tended to be larger and have longer gonopodia and greater gonado- and hepato-somatic indices (GSI and HSI). High GSI in Apopka fish correlated with greater spermatid production, but fewer mature spermatozoa and either the same or lower sperm counts and sperm viability. Taken together, these observations suggest that differentiation of spermatids to spermatozoa is disrupted in Apopka fish, leading to reductions in fertility in some months. Delivery of sperm to females could also be affected in Apopka fish, which exhibit lower prevalence of efferent duct tissue in the testes during the summer.

Effects of Nitrate and Conductivity on Embryo-Larval Fathead Minnows.

Nitrate concentrations have been rising in surface waters over the last century and now frequently exceed drinking water standards and environmental safety benchmarks globally. Health-wise, these trends are concerning because nitrate has been shown to disrupt endocrine function and developmental outcomes. The present study investigated potential sublethal effects of nitrate on developing fathead minnows. Fish were exposed from fertilization through 21 days postfertilization (dpf) to environmentally relevant concentrations of nitrate (0, 2, 5, 10, 25, or 100 mg/L NO3 -N as NaNO3 ). Nitrate effects on hatch timing, heart rate and rhythm at 3 dpf, growth through 21 dpf, swim bladder inflation timing and size, scoliosis, pericardial edema, and mortality were assessed. Because adding NaNO3 increases water conductivity, two conductivity controls were included to match the ionic strength of the 10- and 100-mg/L NO3 -N treatments. Increasing nitrate delayed posterior swim bladder (PSB) inflation in a dose-dependent manner, with possible inhibition of anterior swim bladder (ASB) inflation at higher doses, although nitrate did not affect swim bladder size. Conversely, nitrate did not affect hatch timing or cardiac endpoints at 3 dpf or induce pericardial edema or scoliosis, although there was a noted brood effect on these latter defects. As was observed with increasing nitrate, higher ion concentrations in the conductivity controls caused dose-dependent increases in fish body size at 21 dpf. Increased ionic strength also hastened ASB inflation independently of nitrate. As in other published studies, the observed delay in PSB inflation suggests that nitrate disrupts the thyroid axis and warrants further investigation. In addition, the present study supports the need for conductivity controls in nitrate toxicity studies to distinguish nitrate-specific effects. Environ Toxicol Chem 2023;42:1529-1541. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Toward invasive mussel genetic biocontrol: Approaches, challenges, and perspectives.

Invasive freshwater mussels, such as the zebra (Dreissena polymorpha), quagga (Dreissena rostriformis bugensis), and golden (Limnoperna fortunei) mussel have spread outside their native ranges throughout many regions of the North American, South American, and European continents in recent decades, damaging infrastructure and the environment. This review describes ongoing efforts by multiple groups to develop genetic biocontrol methods for invasive mussels. First, we provide an overview of genetic biocontrol strategies that have been applied in other invasive or pest species. Next, we summarize physical and chemical methods that are currently in use for invasive mussel control. We then describe the multidisciplinary approaches our groups are employing to develop genetic biocontrol tools for invasive mussels. Finally, we discuss the challenges and limitations of applying genetic biocontrol tools to invasive mussels. Collectively, we aim to openly share information and combine expertise to develop practical tools to enable the management of invasive freshwater mussels.

Low oxygen: A (tough) way of life for Okavango fishes.

Botswana's Okavango Delta is a World Heritage Site and biodiverse wilderness. In 2016-2018, following arrival of the annual flood of rainwater from Angola's highlands, and using continuous oxygen logging, we documented profound aquatic hypoxia that persisted for 3.5 to 5 months in the river channel. Within these periods, dissolved oxygen rarely exceeded 3 mg/L and dropped below 0.5 mg/L for up to two weeks at a time. Although these dissolved oxygen levels are low enough to qualify parts of the Delta as a dead zone, the region is a biodiversity hotspot, raising the question of how fish survive. In association with the hypoxia, histological samples, collected from native Oreochromis andersonii (threespot tilapia), Coptodon rendalli (redbreast tilapia), and Oreochromis macrochir (greenhead tilapia), exhibited widespread hepatic and splenic inflammation with marked granulocyte infiltration, melanomacrophage aggregates, and ceroid and hemosiderin accumulations. It is likely that direct tissue hypoxia and polycythemia-related iron deposition caused this pathology. We propose that Okavango cichlids respond to extended natural hypoxia by increasing erythrocyte production, but with significant health costs. Our findings highlight seasonal hypoxia as an important recurring stressor, which may limit fishery resilience in the Okavango as concurrent human impacts rise. Moreover, they illustrate how fish might respond to hypoxia elsewhere in the world, where dead zones are becoming more common.

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory.

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade "Environmental Estrogens" (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ERα) and/or beta (ERß). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-ß-D-galactopyranoside (ONPG) or chlorophenol red-ß-D-galactopyranoside (CPRG)) that are cleaved by ß-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

Environmental exposures and gene regulation in disease etiology.

OBJECTIVE: Health or disease is shaped for all individuals by interactions between their genes and environment. Exactly how the environment changes gene expression and how this can lead to disease are being explored in a fruitful new approach to environmental health research, representative studies of which are reviewed here. DATA SOURCES: We searched Web of Science and references of relevant publications to understand the diversity of gene regulatory mechanisms affected by environmental exposures with disease implications. DATA SYNTHESIS: Pharmaceuticals, pesticides, air pollutants, industrial chemicals, heavy metals, hormones, nutrition, and behavior can change gene expression through a broad array of gene regulatory mechanisms. Mechanisms include regulation of gene translocation, histone modifications, DNA methylation, DNA repair, transcription, RNA stability, alternative RNA splicing, protein degradation, gene copy number, and transposon activation. Furthermore, chemically induced changes in gene regulation are associated with serious and complex human diseases, including cancer, diabetes and obesity, infertility, respiratory diseases, allergies, and neurodegenerative disorders such as Parkinson and Alzheimer diseases. One of the best-studied areas of gene regulation is epigenetics, especially DNA methylation. Our examples of environmentally induced changes in DNA methylation are presented in the context of early development, when methylation patterns are initially laid down. This approach highlights the potential role for altered DNA methylation in fetal origins of adult disease and inheritance of acquired genetic change. CONCLUSIONS: The reviewed studies indicate that genetic predisposition for disease is best predicted in the context of environmental exposures. Second, the genetic mechanisms investigated in these studies offer new avenues for risk assessment research. Finally, we are likely to witness dramatic improvements in human health, and reductions in medical costs, if environmental pollution is decreased.

Reproductive characteristics of male mosquitofish (Gambusia holbrooki) from nitrate-contaminated springs in Florida.

Over the past five decades, anthropogenic nitrate contamination has increased significantly in many ground and surface water systems, creating the need to understand how nitrate impacts the physiology of aquatic animals. We collected adult male mosquitofish (Gambusia holbrooki) from eight springs in Florida with varying nitrate concentrations (0.2-5.1 mg/L NO(3)-N). Fish were evaluated for spring-related differences in body, liver, and gonad size, gonopodium length, muscle testosterone and 11-ketotestosterone (11-KT) concentrations, and sperm counts and viability. Increased nitrate concentration (up to 5 mg/L NO(3)-N) was significantly correlated with decreased total sperm counts per spermatozeugmatum, increased adjusted gonopodium length, and increased adjusted testicular weight. Furthermore, we observed that relatively small differences in spring pH (7.0-7.4) were positively associated with muscle 11-KT concentrations. Finally, minor changes in spring water temperature (21.4-22.9 degrees C) was negatively correlated with adjusted testicular and hepatic weights, and positively correlated with total and live sperm counts. We conclude that male mosquitofish reproduction is not the same among the eight springs tested, and that elevated aquatic nitrate concentrations may explain some of the observed variation.

Alligators, contaminants and steroid hormones.

Steroids are essential for successful reproduction in all vertebrate species. Over the last several decades, extensive research has indicated that exposure to various environmental pollutants can disrupt steroidogenesis and steroid signaling. Although steroidogenesis is regulated by the hypothalamic-pituitary axis, it is also modified by various paracrine and autocrine factors. Furthermore, the classical two-cell model of steroidogenesis in the developing ovarian follicle, involving the granulosa and theca cells in mammals, may not be universal. Instead, birds and probably reptiles use the two thecal compartments (theca interna and theca externa) as sites of steroid production. We have documented that embryonic or juvenile exposure to a complex mixture of contaminants from agricultural and storm water runoff leads to altered steroid hormone profiles in American alligators. Our observations suggest that alterations in plasma steroid hormone concentrations are due in part to altered gene expression, modified hepatic biotransformation and altered gonadal steroidogenesis. Future studies must examine the interplay between endocrine and paracrine regulation in the development and expression of gonadal steroidogenesis in individuals exposed to endocrine disrupting contaminants at various life stages if we are to fully understand potential detrimental outcomes.

Stress and its relation to endocrine function in captive female Siberian sturgeon (Acipenser baeri).

Stress responses to numerous environmental conditions have been studied in a wide range of fish species. Defining the relationship between stress and endocrine function is particularly critical to long-lived species such as sturgeons, whose economic viability relies heavily on proper endocrine function for the production of caviar. In this study, we examined the induced stress response, defined by plasma cortisol and glucose concentrations, and its relationship to plasma 17beta-estradiol, testosterone and 11-ketotestosterone concentrations in cultured female Siberian sturgeon (Acipenser baeri). Fish were acutely stressed using capture and confinement for a period of 4 h, and blood samples were drawn according to four treatment regimens: treatment 1, fish were bled at 0 h; treatment 2, fish were serially bled at 0 h, 1 h and 4 h; treatment 3, fish were bled at 1 h and 4 h; treatment 4, fish were bled at 4 h only. Fish were surgically sexed immediately after drawing blood at 0 h. After 1 h of acute stress, fish demonstrated a marked increase in plasma cortisol concentration, which remained elevated throughout the 4-h sampling period. The plasma concentration of sex steroids did not exhibit an inverse relationship with plasma cortisol concentration. Plasma testosterone concentration was significantly elevated during the periods of greatest stress. Plasma estradiol and 11-ketotestosterone (11-KT) concentrations were not affected by the acute stressors in this experiment. Serial bleedings, however, affected the associated stress response, which is an important consideration for future studies with this species. This is the first study to define the relationship between stress and possible changes in sex steroid concentration in this threatened and commercially important species.

Hormonally active phytochemicals and vertebrate evolution.

Living plants produce a diversity of chemicals that share structural and functional properties with vertebrate hormones. Wildlife species interact with these chemicals either through consumption of plant materials or aquatic exposure. Accumulating evidence shows that exposure to these hormonally active phytochemicals (HAPs) often has consequences for behavior, physiology, and fecundity. These fitness effects suggest there is potential for an evolutionary response by vertebrates to HAPs. Here, we explore the toxicological HAP-vertebrate relationship in an evolutionary framework and discuss the potential for vertebrates to adapt to or even co-opt the effects of plant-derived chemicals that influence fitness. We lay out several hypotheses about HAPs and provide a path forward to test whether plant-derived chemicals influence vertebrate reproduction and evolution. Studies of phytochemicals with direct impacts on vertebrate reproduction provide an obvious and compelling system for studying evolutionary toxicology. Furthermore, an understanding of whether animal populations evolve in response to HAPs could provide insightful context for the study of rapid evolution and how animals cope with chemical agents in the environment.

Water quality influences reproduction in female mosquitofish (Gambusia holbrooki) from eight Florida springs.

Contamination of freshwater ecosystems with nitrate is a growing global concern. Although nitrate pollution is recognized as a cause of aquatic eutrophication, few studies have examined the possible physiological impacts of nitrate exposure. In this study, we surveyed several reproductive variables of viviparous female Gambusia holbrooki (Poeciliidae) captured from eight springs in Florida. The eight springs represent a gradient of nitrate contamination (1-5 mg/L nitrate-nitrogen). We had two objectives in this study: to describe reproductive biology of female mosquitofish in the springs and to understand reproductive variation in the context of water quality, particularly the nitrate concentration. Our data show a significant negative association between nitrate and both dry weight of developing embryos and rate of reproductive activity among mature females. In addition, variation in Gambusia condition index and embryo number and dry weight was related to temperature variation, and hepatic weight was negatively related to dissolved oxygen concentration. Finally, we observed that many of the measured reproductive variables were interrelated and changeable, depending on gestational stage. Specifically, we provide evidence that maternal support of the embryo occurs at least during the first two thirds of gestation and that female fecundity is affected by an apparent tradeoff between embryo size and embryo number.

Environmental context determines nitrate toxicity in Southern toad (Bufo terrestris) tadpoles.

Aquatic nitrate contamination has escalated over the past 50 years, primarily due to intensified fertilizer application and sewage production worldwide. Nitrate's role in the decline of amphibian populations remains unclear, although studies suggest that nitrate exposure affects larval development. We exposed Bufo terrestris tadpoles to environmentally relevant nitrate concentrations from Gosner stage 25 through forelimb emergence. Tadpoles were exposed to fluctuating (0-30 mg/L NO(3)-N, alternated three times per week) or constant nitrate concentrations (0, 5, 15, or 30 mg/L NO(3)-N), and effects were compared in two water types: natural spring water and reverse-osmosis filtered water, fortified with electrolytes (RO(e)). We measured growth and developmental rates, survival, time to metamorphosis, metamorph body size, hepatosomatic index, and whole-body thyroxine (T(4)) concentrations at forelimb emergence. Based on our observations, we reached three main conclusions: (1) in constant nitrate, tadpoles in RO(e) water grew faster, and were generally larger with higher or similar T(4) at metamorphosis than tadpoles raised in spring water, irrespective of nitrate concentration, (2) in fluctuating nitrate (flux), there were no differences in time to or size at metamorphosis in either water type relative to controls; however, mean T(4) concentrations in the flux treatment showed a reversed pattern in the two water types (SP>RO(e)) compared to the general pattern observed with constant nitrate exposure (RO(e)>SP), and (3) the effect of nitrate on growth and development depended on water type. In RO(e) water with high nitrate (RO(e)-30 mg/L NO(3)-N), tadpoles metamorphosed an average of 5 days (13%) earlier than control animals, but were similar in size to controls. However, spring water tadpoles reared in high nitrate (30 mg/L) delayed metamorphosis by 7 days (18%) compared to animals reared in SP-0, and 11 days (32%) compared to tadpoles raised in RO(e)-30. This delayed development allowed SP-30 tadpoles to reach a larger size that was more similar to metamorphs raised in RO(e) water. Based on information from other studies, we conclude that, in RO(e) water, tadpoles exhibited an expected stress response to nitrate (e.g. metamorphosed earlier as nitrate concentration increased). However, we suggest that, in spring water, tadpoles were exposed to additional stressors that decreased growth rate and thyroxine concentrations, and that this effect was modified by nitrate.

Disturbed sexual characteristics in male mosquitofish (Gambusia holbrooki) from a lake contaminated with endocrine disruptors.

Previous laboratory studies have demonstrated that estrogenic and antiandrogenic chemicals can alter several sexual characteristics in male poeciliid fishes. Whether similar disturbances occur under field conditions remains to be confirmed. Lake Apopka, Florida, is contaminated with numerous chemicals, some of which possess endocrine-disrupting activity. Male mosquitofish (Gambusia holbrooki) were collected monthly from December 2000 through May 2001 from Lake Apopka and two nearby reference lakes, Orange Lake and Lake Woodruff National Wildlife Refuge. Selected sexual characteristics were compared temporally and among lakes during the collection period. Male fish from Lake Apopka had slightly shorter gonopodia and on average 32 and 47% fewer sperm cells per milligram testis, when compared with the fish collected from Orange Lake and Lake Woodruff, respectively. The testes weights increased markedly during spring, with significantly smaller testes in fish from Lake Apopka than from Orange Lake, but surprisingly, the smallest testes occurred in males obtained from the Lake Woodruff population. The highest liver weights were found in the Lake Apopka population. Whole-body concentrations of testosterone and estradiol varied among months; the peak testosterone concentration occurred in January and was significantly lower in male fish from Lake Apopka compared with Orange Lake. The intensity of male courtship behavior was highly correlated to body testosterone concentration, but no statistically significant differences in sexual behavior among the lakes were found. We conclude that sexual characteristics of relevance to male reproductive capacity are altered in the Lake Apopka mosquitofish population, and we discuss the presence of chemicals with antiandrogenic effects in Lake Apopka as a possible cause of the observed alterations.

Variation in sex steroids and phallus size in juvenile American alligators (Alligator mississippiensis) collected from 3 sites within the Kissimmee-Everglades drainage in Florida (USA).

This 3-year study was designed to examine variation in plasma sex steroids, phallus size, and the standard error (S.E.) associated with these endpoints in juvenile alligators collected from 3 sites within the Kissimmee-Everglades drainage (Florida, USA) with varying concentrations of sediment organochlorine contaminants. We hypothesized that decreased plasma sex steroid concentrations and phallus size would be observed in the higher contaminant site when compared to the intermediate and lower contaminant sites. Furthermore, we hypothesized that greater S.E. associated with these endpoints would be observed for the populations from more contaminated sites. We found that differences existed with females from the higher contaminant site exhibiting lower plasma estradiol-17beta (E2) and testosterone (T) concentrations. Males from the higher contaminant site exhibited smaller phallus sizes than males from the intermediate and lower contaminant sites. Smaller phallus size in this case differed from that reported in Lake Apopka male alligators [Gen. Comp. Endocrinol. 116 (1999) 356] in that a significant positive relationship between body size and phallus size existed. No difference among sites was observed in plasma T for males. Lower S.E. was associated with E2 and T concentrations in females from the higher contaminant site and in phallus size in males from the higher contaminant site. This pattern was opposite to what we had hypothesized. We concluded that variation in plasma E2 and T concentrations, phallus size, and the S.E. associated with these endpoints exists among the 3 sites with the patterns matching the patterns of organochlorine contamination, although S.E. patterns were opposite to what was predicted.